Glucosylation of cytokinin analogues

Glucosylation of adenine and 6-methylaminopurine was not detected in derooted 10-day-old radish seedlings. However, 4-(purin-6-ylamino)butanoic amide and 6-(3,4-dimethoxybenzylamino)purine (N⁶-substituted adenines with negligible cytokinin activity), like the highly active cytokinin 6-benzylaminopurine, were converted to 7-glucopyranosides. The N²-substituted guanine, 2-benzylaminopurin-6-one, and 6-benzylamino-2-(2-hydroxy-ethylamino)purine were also metabolized to glucosides which were probably 7-glucopyranosides. Hence glucosylation of purines is not restricted to N⁶-substituted adenines with strong cytokinin activity. Although only ca 1.6% of 6-benzylamino-9-(4-chlorobutyl)purine taken up by the derooted seedlings could be accounted for as 7- and 9-glucosides, a considerable proportion was metabolized to these glucosides in cotyledons excised from 2-day-old radish seedlings. The high cytokinin activity of this 9-substituted compound may be a consequence of cleavage of the 4-chlorobutyl groud at N-9.     

Binding site of novel 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5- triazine herbicides in the D1 protein of Photosystem II

A series of replacement experiments of [¹⁴C]-triazines, [¹⁴C]-atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [¹⁴C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [¹⁴C]-Atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [¹⁴C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolic activation of hexamethylmelamine and pentamethylmelamine by liver microsomal preparations

Incubation of [¹⁴C]-ring labeled hexamethylmelamine and pentamethylmelamine with rat and mouse liver microsomal preparations results in metabolic activation of both drugs as measured by covalent binding of radiolabel to acid-precipitable microsomal macromolecules. Covalent binding is dependent on viable microsomes, NADPH, and molecular oxygen. Binding of HMM (280 pmol/mg protein/15 min) was approximately 5 times greater than that observed for PMM (60 pmol/mg protein/15 min), and represents 0.22% of incubated material. Similar results were found with [¹⁴C]-methyl labeled substrates. Pretreatment with phenobarbital increased covalent binding while addition of SKF 525-A, addition of glutathione, or incubation in an 80% carbon monoxide atmosphere reduced covalent binding.     

Activity and metabolism of 9-substituted cytokinins during lettuce seed germination

The activities of N⁶-benzyladenine (BA) and its 9-substituted methyl, methoxymethyl, tetrahydropyranyl, cyclopentyl, and cyclohexyl analogs were determined for the promotion of lettuce seed (Lactuca sativa L. cv. Grand Rapids) germination. Cytokinin concentrations used were 10^?4, 10^?5, 10^?6 and 10^?7M. All seeds were incubated under total dark conditions at 28 ± 1°C. After 48 h the percentage of germination was recorded. A comparison of means based on Duncan's Multiple Range Test allowed for a ranking of cytokinin activities for the promotion of lettuce seed germination. The activities were: BA = 9-tetrahydropyranyl BA > 9-methyl BA > 9-methoxymethyl BA > 9-cyclopentyl BA > 9-cyclohexyl BA. The results were significant at the 95% confidence level as determined by analysis of variance. In order to study the metabolism of a cytokinin, lettuce seeds were incubated with 9-methyl-BA-methylene-¹⁴C. The labeled cytokinin was prepared by refluxing benzylamine hydrochloride (methylene-¹⁴C) with an equal molar ratio of 6-chloro-9-methylpurine. Final cytokinin concentration was 10^?5M. Incubation periods were 2, 4, 8, 12, 16 and 20 h at 28 ± 1°C under total dark conditions. At the end of the various time periods the seeds were extracted with 70 percent methanol. The resulting extracts were purified and radioactive metabolites identified by solvent fractionation, Sephadex LH-20 column chromatography, and paper chromatography. Co-chromatography with authentic standards in the appropriate solvent system revealed that the metabolites were 9-methyl BA, N⁶-benzyladenosine-5′-monophosphate, and N⁶-benzyladenosine. The results lend support to the theory that the cytokinin ribonucleotide serves as a storage form which is converted to the active ribonucleoside as needed during lettuce seed germination.     

Mapping the carbon reduction cycle: a personal retrospective

The photosynthetic carbon reduction cycle was elucidated through the use of ¹⁴CO₂ during photosynthesis to label metabolic intermediates. Mapping and proof of the cycle required identification of labeled metabolites, observation of changes in levels of labeled metabolites during transitions from light to dark and from high to low CO₂ levels, determination of intramolecular distribution of ¹⁴C within the metabolites after a few seconds of photosynthesis with ¹⁴CO₂, and estimation of metabolite concentrations, used to calculate true free energy changes at each step in the cycle. This revised version was published online in August 2006 with corrections to the Cover Date.     

On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

The stereochemistry of cyclization in abscisic acid

The hydroxylation of the pro-6′-(R)-methyl of (+)-abscisic acid, which then cyclises to phaseic acid, was used to define the origin in mevalonate of the 6′-methyl groups. Abscisic acid (ABA), biosynthesised from [2-¹⁴C, 2-³H₂]-mevalonate, was metabolized to phaseic acid by tomato shoots. The slight loss of [³H] from the phaseate, and to a lesser extent from the ABA, suggested that the unlabelled 6′-methyl was hydroxylated. This was confirmed by Kuhn-Roth oxidation of methyl phaseate to give [¹⁴C, ³H]-acetate. The data also suggest that ABA is converted to dihydrophaseate via free phaseate, the conjugates being formed from each free acid.     

7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N²-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N²-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram− bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10 μg/ml), and were inactive against the Gram− organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.     

LINKING TIME‐DEPENDENT CARBON‐FIXATION EFFICIENCIES IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) TO UNDERLYING METABOLIC PATHWAYS1

The chl‐specific short‐term ¹⁴C‐based production (P^b) measurement is a widely used tool to understand phytoplankton responses to environmental stresses. However, among the metabolic consequences of these stresses is variability in lifetimes of newly fixed carbon that cause P^b to range between chl‐specific net primary production (NPP*) and chl‐specific gross photosynthetic electron flow that is available for carbon reduction () depending on growth rate. To investigate the basis for this discrepancy, photosynthate utilization was characterized in Dunaliella tertiolecta Butcher grown at three different growth rates in N‐limited chemostats. P^b was measured throughout a 2 min to 24 h time course and showed clear growth‐rate‐dependent differences in lifetimes of newly fixed carbon. ¹⁴C pulse‐chase experiments revealed differences in patterns of carbon utilization between growth rates. At high growth rate, the majority of ¹⁴C was initially fixed into polysaccharide and lipid, but the relative contribution of each labeled biochemical pool to the total label changed over 24 h. In fast‐growing cells, labeled polysaccharides decreased 50%, while labeled lipids increased over the first 4 h. At low growth rate, ¹⁴C was initially incorporated primarily into protein, but the contribution of labeled protein to the total label increased over the next 24 h. Together, time‐resolved measurements of P^b and cellular NAD and NADP content suggest an enhanced role for alternative dissipation pathways at very low growth rate. Findings of this study contribute to an integrated understanding of growth‐rate‐dependent shifts in metabolic processes from photosynthesis to net growth.     

Determination by Gas Chromatography-Mass Spectrometry of [N(5)]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [¹⁵N₅]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium (²H)-labeled internal standards. Incorporation levels of the [¹⁵N₅]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5′-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-¹⁴C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-¹⁴C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of ¹⁴C incorporation into cytokinins declined.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

A study of the hypothetical role of cytokinins in completion of tRNA

Summary It has been suggested that the effect of cytokinins in retarding leaf senescence comes about through their incorporation into tRNA. To test this hypothesis, kinetin-8-¹⁴C, 6-benzylaminopurine-benzyl-7-¹⁴C and adenine-8-³H were applied to detached tobacco leaves, and the nucleic acids were thereafter extracted and chromatographed on MAK columns. Kinetin-8-¹⁴C and adenine-8-³H were readily incorporated into RNA in a similar pattern. 6-Benzylaminopurine-benzyl-7-¹⁴C was effective in delaying chlorophyll loss but was not incorporated into any nucleic-acid fraction. It is concluded that the possibility of cytokinins retarding leaf senescence by completion of tRNA is not supported.     

Biochemical effects of miconazole on fungi. II. Inhibition of ergosterol biosynthesis in Candida albicans.

The effects of the antifungal agent miconazole nitrate on the ergosterol biosynthesis in Candida albicans were investigated after in vitro contact with the drug for 1, 4, 16 and 24 h. A time- and dose-(2.10^?10–10^?4 M) dependent inhibition of [¹⁴C]acetate incorporation into ergosterol was observed. Fifty percent inhibition of the acetate incorporation into ergosterol was found after 1 h incubation in the presence of 10^?9 M miconazole. Simultaneously 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 4,14-dimethylzymosterol and 14-methylfecosterol accumulated.The accumulation of 14 α-methyl sterols suggests that this antifungal agent is a potent inhibitor of one of the metabolic steps involved in the demethylation at C-14. The absence of 24-methyl sterols and of sterols with a C-22 [23] double bond in miconazole treated C. albicans indicates that miconazole also inteferes with the reduction of the 24(28)-double bond and with the introduction of the 22(23)-double bond.Miconazole also intervenes to a small extent in triglyceride synthesis. However, in all circumstances studied, ergosterol biosynthesis was affected at lower doses than those interfering with the acetate incorporation into triglycerides. 16 and 24 h of incubation in the presence of miconazole (≥ 10^?6 M) also resulted in an increased fatty acid synthesis.It is suggested that the miconazole-induced inhibition of the C-14 demethylation may be at the origin of the previously observed permeability changes in miconazole treated C. albicans.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Cytokinin biosynthesis in crown-gall tissue ofVinca rosea

When care was taken to minimise the effects of phosphatase activity during extraction ofVinca rosea crown-gall tumour tissue, a large proportion of extractable cytolinin activity was present in the nucleotide fraction. Analysis using ion-exchange chromatography followed by enzymic or chemical degradation and subsequent identification of the biologically active material indicated that this activity was due to zeatin riboside 5′-monophosphate. This was also the major radiolabelled cytokinin formed when this tissue was supplied with [¹⁴C]adenine. The incorporation of radioactivity from [¹⁴C]adenosine into free cytokinins was also shown, but no incorporation of radioactivity was found when [³H]mevalonic acid lactone was supplied to this tissue under the same conditions. In parallel experiments using normal stem callus tissue ofV. rosea, no incorporation of [¹⁴C]adenine into free cytokinins was observed. The significance of these results is discussed in relation to a possible transfer-RNA-independent pathway of cytokinin biosynthesis, operating primarily at the mononucleotide level.     

Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis

The efficiency with which developing maize embryos convert substrates into seed storage reserves was determined to be 57–71%, by incubating developing maize embryos with uniformly labeled ¹⁴C substrates and measuring their conversion to CO₂ and biomass products. To map the pattern of metabolic fluxes underlying this efficiency, maize embryos were labeled to isotopic steady state using a combination of labeled ¹³C-substrates. Intermediary metabolic fluxes were estimated by computer-aided modeling of the central metabolic network using the labeling data collected by NMR and GC-MS and the biomass composition. The resultant flux map reveals that even though 36% of the entering carbon goes through the oxidative pentose-phosphate pathway, this does not fully meet the NADPH demands for fatty acid synthesis. Metabolic flux analysis and enzyme activities highlight the importance of plastidic NADP-dependent malic enzyme, which provides one-third of the carbon and NADPH required for fatty acid synthesis in developing maize embryos.